Retinal Ganglion Cell Axon Response to Guidance Molecules
Stephen F Oster and David W Sretavan
Department of Ophthalmology, University of California, San
Francisco
Correspondence to: David W Sretavan, MD, PhD, Department of Ophthalmology, K107, Box 0730, University of California San Francisco,
10 Kirkham Street, San Francisco, CA 94143,
USA Email: dws@itsa.ucsf.edu
Accepted for publication: 1st March 2003
Promotion of axon elongation –These growth cone tipped axons are extending on growth-promoting guidance molecules (the transmembrane cell adhesion molecule L1) coated to form a substratum on the bottom of the culture dish. Note the filopodial and lamellapodial movement, and fan-shaped morphology, characteristic of active growth cones. Growth cone collapse –This growth cone was extending on a growth-promoting substratum for a period of time, and was then exposed to an inhibitory axon guidance moleculeEphB2. This molecule caused the collapse of the growth cone and retraction of the axon. |
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Background
The specific patterns of neural connectivity essential for visual information processing are formed during development in a process known as axon pathfinding. Pathfinding is orchestrated by axon guidance molecules, which are deployed in the embryonic environment to steer elongating axons. Developing axons interact with guidance molecules at a morphological specialization at their tip called the growth cone. Growth cones are motile, exploratory structures, and their surfaces contain a large number of receptor molecules that sense and respond to axon guidance molecules. Although many axon guidance molecules exist, their effects on developing axons and growth cones can generally be divided into two types. Some guidance molecules promote the growth of axons and often attract growth cones to extend towards a source of the molecule. Other molecules have an opposite effect and inhibit axon growth by repelling axons or causing the growth cone to stop or collapse. (See Oster, SF, Sretavan, DW. Connecting the Eye to the Brain: The Molecular Basis of Ganglion Cell Axon Guidance. Brit J Ophthalmol 2003; 87:639-645 http://bjo.bmjjournals.com/cgi/content/full/87/5/639).
Videos and Comments
Promotion of axon elongation – These embryonic mouse retinal axon growths are extending on a guidance molecule that has been coated to form a substratum on the bottom of the culture dish. Note the characteristic fan-shaped morphology of the growth cone and movements of the growth cone filopodia and lamellapodia. In this video, axons are growing on the guidance molecule L1, a transmembrane cell adhesion molecule expressed by retina ganglion cell axons during their pathfinding journey. In vivo, L1 binds to L1 molecules on other retinal axons through homophilic interactions. This ability of retinal axons to bind to one another, or fasciculate, is important during the exit of ganglion cells axons from the retina into the optic nerve.
Growth cone collapse – This growth cone was extending on a growth-promoting substratum for a period of time, and was then exposed to an inhibitory axon guidance molecule. The growth cone responds by undergoing a collapse, during which it loses filopodial and lamellapodial structures and ultimately exhibit a retraction of the retinal axon. The inhibitory molecule used in this case was EphB2, a guidance molecule that is normally expressed in multiple parts of the developing visual system, including the developing retina and the superior colliculus. The ability of EphB proteins to influence the growth of retinal axons is critical for the orderly growth of retinal axons to the optic disc and the formation of the retinotopic map in the midbrain.
Techniques
Retinal tissue was obtained from E14 mouse embryos harvested from anesthetized timed pregnant C57/B6 mice. Embryonic retinas were cut into pieces, and explanted overnight at 370C, 55 CO2 in F12 medium and N2 supplement (F12/N2 medium) (Gibco). Explants were cultured in 35 mm coverslip dishes (MakTek), which were pre-coated with either 5 µg/ml L1-Fc (Video #1) or 5 µg/ml laminin (Video #2) as a growth substratum. After overnight culture, coverslip dishes containing retinal explants were overlaid with pre-warmed mineral oil (Sigma) and maintained at 370 C on a microscope stage incubator with CO2 influx. Time-lapse images of growth cones were captured at 1 minute intervals with a CCD camera (PXL2, Photometrics) using Hoffman optics and Deltavision image acquisition software (API). For Video #2, EphB2-Fc was applied using a glass micropipette loaded with 50 µg/ml EphB2-Fc that was used to deliver the protein near the growth cone by repeated pressure pulses via a Picospritzer (General Valve).
Acknowledgement
We thank Eric Birgbauer and Leejee Suh for help with experiments and reagent construction.
References